The aim of this research was to determine the effect of using phytase, carbohydrases (NSPases), and protease in a diet for juvenile rainbow trout Oncorhynchus mykiss on their intestinal histology.
Seven isoproteic (42% CP) and isocaloric (20% EE) experimental diets were formulated to meet the nutritional requirements of rainbow trout (NRC, 2011) and evaluated under a completely randomized design. Soy-derived protein ingredients made up 30% of the final formulation, with 25% corn gluten and only 10% fishmeal. The diets were identified as follows: C, Control: No exogenous commercial enzyme addition; M: Diet C + β-mannanase (100,000 BMU kg-1); F: Diet C + Phytase (4,000 FTU kg-1); FM: Diet F + β-mannanase (100,000 BMU kg-1); FMX: Diet FM + Xylanase (4,000 XU kg-1); FMXG: Diet FMX + β-glucanase (500 BGU kg-1); FMXAP: Diet FMX + Amylase (400 AU kg-1) + Protease (8,000 PU kg-1). The experiment involved 25 fish tank-1. It was carried out in 21 tanks of 300 L under a RAS for 40 days. Five fish per tank were desensitized and then euthanized with an overdose of benzocaine. All procedures were performed in accordance with the reference guidelines of the Guide for the Care and Use of Laboratory Animals. Fish surface was disinfected with 70% alcohol and dissected using sterile instruments. One cm midgut segments were fixed in Bouin’s solution, then dehydrated using a graded alcohol and xylene series. The samples were paraffin-embedded, sectioned at 5 μm, and stained with hematoxylin-eosin (HE). Finally, photographs of the sections were taken with a Zeiss microscope and camera and processed using ImageJ software. The fold surface area [mm2] was calculated using the following formula: (2π) × (Fold Widht/2) × (Fold Lenght)/106. Once the assumptions of normality and homoscedasticity were verified, the data underwent an analysis of variance (ANOVA).
Results are shown in Table 1. Although a one-way ANOVA revealed no significant differences for any of the determinations, a two-way analysis conducted on the first four diets revealed significant main effects of β-mannanase inclusion (M diet), reducing fold length and surface area (P-values = 0.0188 and 0.0039, respectively).
Histopathological analysis of HE-stained sections revealed overall good tissue preservation in all treatments. Goblet cell counts in diet M were elevated, suggesting some degree of compensatory response to reduced fold surface area. Conversely, for the FMXG diet, this could be linked to findings at ontogenetically older fish fed a higher-energy diet, consistent with a potential increase in lipid availability due to specific added enzymatic activity. The inclusion of NSP via the plant-based ingredients and added enzymes did not induce changes in the histometric parameters evaluated. Consequently, the beneficial or detrimental physiological effects of NSP in fish remain controversial and dose-dependent. An improvement in intestinal morphology due to protease was not observed, although protease was not evaluated individually.